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Analysis Strategy and Research Method for N-linked Intact Glycopeptide by Mass Spectrometry (I)

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As one of the most common and important protein modification methods, protein glycosylation has always been the focus. In the past years, the commonly used method of N-linked glycoproteomics research is to analyze released glycans or de-glycosylated peptides separately. This strategy reduces the difficulty of analysis, while it also lost glycosite-specific glycosylation information. In recent years, mass spectrometry strategies and methods for intact glycopeptides have been gradually established. Generally, to achieve the identification and quantification of intact glycopeptides, the first step is to enrich glycopeptides from complex samples to reduce the affects from non-glycosylated peptides, then the mass spectrometry parameter settings need to be adjusted to satisfy the fragment features of glycopeptides, importantly the related software also need to be developed for the precise identification of the peptide sequence and glycan structures or compositions of the intact glycopeptides. These three main aspects of the strategies for mass spectrometry-based intact glycopeptide analysis are discussed in this article.

1. Enrichment strategy for intact glycopeptides

In glycoproteomics research, the research object is usually complex biological sample. The dynamic range of protein abundance in the sample varies greatly. At the same time, many glycoproteins have low abundance. All or specific glycoprotein groups are studied. Enrichment will help to identify more glycoproteins. In addition, because glycosylated peptides often only account for 2% to 5% of protein digested peptides, the signal during mass spectrometry analysis is easy to be inhibited by non-glycopeptides, so the primary problem faced by glycoproteomics research is the effective separation and enrichment of glycoproteins / glycopeptides, that is, the removal of non-glycoproteins / non-glycopeptides.

The methods currently available for the enrichment of intact glycopeptides include hydrophilic interaction liquid chromatography (HILIC), lectin affinity and boric acid affinity, etc. HILIC is based on the strong hydrophilic property of the polyhydroxy-rich sugar chain structure, which enables the separation of glycopeptides and non-glycosylated peptides, and can maintain the integrity of the sugar chain structure, both for N-glycopeptides and for O-glycopeptide. However, due to the weak interaction between the sugar chain and the stationary phase, glycopeptides that only link monosaccharides or oligosaccharides (such as O-glycopeptides) are often not well enriched by HILIC. Lectins are a class of sugar-binding proteins that can specifically recognize and bind to specific glycosyl sequences in structure-specific monosaccharides or glycans. Their binding to sugar chains is non-covalent and reversible. After the glycoprotein or glycopeptide is captured by the lectin, the glycoprotein or glycopeptide is usually eluted with a specific monosaccharide by competitively binding the lectin, thereby achieving enrichment of the glycoprotein.

2. Intact glycopeptides analysis by mass spectrometry

2.1. Intact glycopeptide in MS1

During the mass spectrometry analysis, due to the high complexity of biological protein samples, the samples enriched with glycopeptides are usually still doped with many non-glycosylated peptides, which often interfere with the mass spectrometry of intact glycopeptides. At present, there are some simple and effective methods to screen the mass spectrum peaks of intact glycopeptides from the spectrum of first stage of mass spectrometry (MS1). These primary screening can effectively reduce the mass spectrum of non-glycosylated peptides. Analyze time to increase the opportunity for the identification of intact glycopeptides. The methods reported include:

2.1.1. Direct identification of molecular mass

The fundamental basis is that the concept of molecular mass is determined by defining the molecular mass of 12C as 12, and the molecular mass of other elements is determined by comparing with the molecular mass of 12C. Elements can be divided into exactly positive integers according to mass (Such as 12C, 12.00000), or greater than a positive integer (such as 1H, 1.00783; 14N, 14.00307) or less than a positive integer (such as 16O, 15.99491; 32S, 31.97207). Usually in peptides, N and H content is the highest, their actual mass is slightly larger than the molecular weight after rounding, and in glycopeptides, O and S content is relatively high, their actual mass is slightly smaller than the molecular weight after rounding. So for the molecule Glycopeptides of equal mass and ordinary polypeptides without glycosylation, the molecular mass of glycopeptides is usually smaller than polypeptides. This feature can be used to distinguish glycopeptides from ordinary polypeptides. Froehlich et al. used this method to simulate experiments, and got a simple classifier that can distinguish the glycopeptide precursor ion from the peptide precursor ion, and divide the integer part and fraction part of the mass of the glycopeptide / peptide into equations to classify the glycopeptide. In the mass spectrometry mass accuracy is 10ppm, the method can identify intact glycopeptides with a sensitivity of more than 89% and a specificity of more than 93%. With the emergence and popularity of higher resolution and mass accuracy mass spectrometry, it can identify intact glycopeptide. The specificity of the peptide will be further enhanced. However, this method requires that the sugar chain portion of the glycopeptide occupies a large molecular mass ratio, that is, the content of oxygen atoms (O) in the glycopeptide is high, which is more distinguishable from ordinary peptides.

2.1.2. Isotope distribution identification

Because most intact glycopeptides (especially N-glycopeptides) contain more oxygen (O) than non-glycosylated polypeptides, and the isotope of the O element in nature contains 2u difference between 16O and 18O, which is different from the peptide Other major elements C, H, N, etc. (there is only 1u difference between isotopes). Therefore, intact glycopeptides and ordinary peptides usually have different isotope abundance distributions, and their characteristic isotope peak patterns can theoretically be used to identify MS1 Whether the detected molecule is a glycopeptide precursor ion peak. However, the premise of the application of this method is to detect accurate isotope abundance, which is rarely used alone in actual applications. However, the direct identification of molecular mass is used in conjunction with the isotope abundance. Combined with high-resolution and high-precision mass spectrometry, it is theoretically feasible to identify intact glycopeptides at the level of the MS1 precursor ion. Direct identification of the intact glycopeptide at the MS1 level, and then selective MS2 analysis can increase the glycopeptide precursor ion to achieve the purpose of maximum analysis and identification of intact glycopeptides.

To be continued in Part II…

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